In vitro regeneration of Vigna unguiculata (L.) Walp. cv. Blackeye cowpea via shoot organogenesis

An in vitro regeneration system was developed in cowpea [Vigna unguiculata (L.) Walp.] Blackeye. Among several explants studied, shoot initiation response was observed from shoot apices of 3–5-day-old seedlings. The optimal medium for maximum shoot initiation comprised MS salts, B₅ vitamins, 8.88 μM N ⁶-benzylaminopurine, 1 gl^-1 casein hydrolysate, 342 μM L-glutamine, 3% sucrose, 0.3% phytagel, adjusted to pH 5.8. A shift in pH from 5.8 to 7.0 had no effect on shoot initiation and on number of shoots per explant. The highest shoot initiation frequency (77%) was obtained using this preferred medium, reaching a maximum of eight shoots per explant. For shoot elongation, 14 μM gibberellic acid was supplemented in the shoot initiation medium. Presence of indolebutyric acid in the rooting medium had no effect on root induction. The regenerated plants were fertile and developed normally.     

The F‐box protein HAWAIIAN SKIRT is required for mimicry target‐induced microRNA degradation in Arabidopsis

Mimicry target-directed micro RNA degradation is widespread and highly conserved among eukaryotes. However, little is known about its mechanism of action. In this letter, by using STTM160(target mimic of mi R160) as a reporter, we show that dysfunction of HAWAIIAN SKIRT(HWS) suppresses the pleiotropic phenotype of STTM160. Small RNA sequencing and Northern blot analyses suggested that HWS only affects a subset of micro RNAs. Intriguingly,we identified a stable coexistence of mi R160/mi R399 and their mimicry targets within the AGO1 complex when HWS is compromised, pointing to a possible role of HWS in the clearance of RNA-induced silencing complexes associated with mimicry target.     

Fast protein sequencing of monoclonal antibody by real-time digestion on emitter during nanoelectrospray

Growth in the pharmaceutical industry has led to an increasing demand for rapid characterization of therapeutic monoclonal antibodies. The current methods for antibody sequence confirmation (e.g., N-terminal Edman sequencing and traditional peptide mapping methods) are not sufficient; thus, we developed a fast method for sequencing recombinant monoclonal antibodies using a novel digestion-on-emitter technology. Using this method, a monoclonal antibody can be denatured, reduced, digested, and sequenced in less than an hour. High throughput and satisfactory protein sequence coverage were achieved by using a non-specific protease from Aspergillus saitoi, protease XIII, to digest the denatured and reduced monoclonal antibody on an electrospray emitter, while electrospray high voltage was applied to the digestion mixture through the emitter. Tandem mass spectrometry data was acquired over the course of enzyme digestion, generating similar information compared to standard peptide mapping experiments in much less time. We demonstrated that this fast protein sequencing method provided sufficient sequence information for bovine serum albumin and two commercially available monoclonal antibodies, mouse IgG1 MOPC21 and humanized IgG1 NISTmAb. For two monoclonal antibodies, we obtained sequence coverage of 90.5–95.1% for the heavy chains and 98.6–99.1% for the light chains. We found that on-emitter digestion by protease XIII generated peptides of various lengths during the digestion process, which was critical for achieving sufficient sequence coverage. Moreover, we discovered that the enzyme-to-substrate ratio was an important parameter that affects protein sequence coverage. Due to its highly automatable and efficient design, our method offers a major advantage over N-terminal Edman sequencing and traditional peptide mapping methods in the identification of protein sequence, and is capable of meeting an ever-increasing demand for monoclonal antibody sequence confirmation in the biopharmaceutical industry.     

Exogenous spermidine improves the sucrose metabolism of lettuce to resist high-temperature stress

Plant Growth Regulation - Lettuce is a popular fresh vegetable, and high-temperature stress will reduce the yield of lettuce. Spermidine is an essential phytohormone in plant stress responses....     

Development of molecular markers based on CRa gene sequencing of different clubroot disease-resistant cultivars of Chinese cabbage

Background

CRa is a key gene in Chinese cabbage (Brassica rapa ssp. pekinensis) that confers resistance to Plasmodiophora brassicae. In order to efficiently screen the clubroot resistance (CR) gene CRa in breeding, two functional codominant markers of the CRa gene were developed.

Methods and results

In this study, through comparing the CRa allele sequences in resistant and susceptible cultivars of Chinese cabbage, we found two insertion and deletion of sequence variations in the fourth exon between resistant and susceptible cultivars. Two functional codominant markers for CRa gene were obtained based on the variations, namely, CRaEX04-1 and CRaEX04-3. The lengths of the extended fragment of CRaEX04-1 marker were 321 bp and 186 bp in resistant and susceptible cultivars, respectively. In contrast, those of CRaEX04-3 were 704 bp and 413 bp, respectively. We verified the genetic stability between the developed markers and CRa gene using 57 Chinese cabbage cultivars with known resistance and two genetic populations. The results showed that the marker identification was completely consistent with the known phenotypes in 57 cultivars. The marker identification results followed the 3:1 of Mendel’s first law in the F₂ population, and the 1:1 of Mendel’s first law in the BC₁.

Conclusions

CRaEX04-1 and CRaEX04-3 can be used as a practical molecular marker for breeding and germplasm resource creation of clubroot disease-resistant Chinese cabbage.

     

The Xenopus tropicalis orthologue of TRPV3 is heat sensitive

Thermosensitive members of the transient receptor potential (TRP) family of ion channels (thermal TRP channels) play a crucial role in mammalian temperature sensing. Orthologues of these channels are present in lower vertebrates and, remarkably, some thermal TRP orthologues from different species appear to mediate opposing responses to temperature. For example, whereas the mammalian TRPV3 channel is activated by heat, frog TRPV3 is reportedly activated by cold. Intrigued by the potential implications of these opposing responses to temperature for the mechanism of temperature-dependent gating, we cloned Xenopus laevis TRPV3 and functionally expressed it in both mammalian cell lines and Xenopus oocytes. We found that, when expressed in mammalian cells, the recombinant channel lacks the reported cold sensitivity; rather, it is activated by temperatures >50°C. Furthermore, when expressed in mammalian cells, the frog orthologue shows other features characteristic of mammalian TRPV3, including activation by the agonist 2-aminoethoxydiphenyl borate and an increased response with repeated stimulation. We detected both heat- and cold-activated currents in Xenopus oocytes expressing the recombinant frog TRPV3 channel. However, cold-activated currents were also apparent in control oocytes lacking recombinant TRPV3. Our data indicate that frog TRPV3 resembles its mammalian orthologues in terms of its thermosensitivity and is intrinsically activated by heat. Thus, all known vanilloid receptors are activated by heat. Our data also show that Xenopus oocytes contain endogenous receptors that are activated by cold, and suggest that cold sensitivity of TRP channels established using Xenopus oocytes as a functional expression system may need to be revisited.